JP2003235556A - Antihuman chymase monoclonal antibody and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antihuman chymase monoclonal antibody capable of assaying the human chymase and having high specificity, a method for assaying the human chymase, etc., utilizing the antibody and a technique of testing and diagnosing diseases associated with the human chymase. <P>SOLUTION: The antihuman chymase monoclonal antibody to a proteolytic product obtained by enzymically treating the human chymase is provided. The antihuman chymase monoclonal antibody to a recombinant human chymase prepared by utilizing an insect living body of the family Bombyx or the family Noctuidae or a cultured cell of the insect as a host is obtained. The method for assaying the human chymase comprises utilizing the antibodies. An assaying kit and a diagnostic agent for hypertension, allergic diseases or circulatory diseases utilize the antibody. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to anti-human chymase antibodies and their use, and more specifically, to vascular lesions associated with angiotensin II produced by human chymase (recompression after percutaneous coronary angioplasty, Intimal cavity thickness after bypass grafting), hypertension, cardiac hypertrophy, myocardial infarction, arteriosclerosis, cardiovascular diseases such as diabetic and non-diabetic renal diseases, diseases such as solid tumors, and allergies associated with chymase Anti-human chymase antibody that enables the development of clinical test reagents for the diagnosis of pathological conditions of diseases such as rheumatoid arthritis, keloids, psoriasis, and atopic dermatitis, prognosis and prognosis of treatment, and clinical evaluation of chymase inhibitors The present invention also relates to a method for measuring human chymase, a kit for measuring human chymase, and the like using the same.

[0002]

2. Description of the Related Art In cardiovascular diseases and inflammatory diseases,
Angiotensin II, which is a bioactive peptide
sin II: abbreviated as “Ang II” hereinafter) plays the central role. Angiotensin converting enzyme (Angiotensin Co) is used as an enzyme involved in the AngII production mechanism.
nverting Enzyme: hereinafter abbreviated as "ACE") and chymase are known. Until now, it was considered that ACE was mainly responsible for the production of AngII in vivo, and research on this was progressing. However, in recent years, chymase has been proved to produce AngII independently of this ACE, and chymase and cardiovascular disease,
I became very interested in the pathophysiology of inflammatory diseases (Miyazaki et al., “Hormones and clinical practice”, No.42, p.
p1145-1152 (1994).).

The pathological conditions caused by the abnormal elevation of AngII include vascular lesions (restenosis after percutaneous coronary angioplasty (PTCA), intimal thickening after bypass grafting), hypertension,
Cardiovascular diseases such as cardiac hypertrophy, myocardial infarction, arteriosclerosis, diabetic and non-diabetic renal diseases (Miyazaki Mizuo, "Cardiology",
No.41, vol.1 pp115-121 (1997).) And solid tumors and allergic diseases (eg, rheumatoid arthritis, keloids, psoriasis, atopic dermatitis), which are exposed from mast cells. It is thought to be due to the action of human chymase. Therefore, it is expected that serum chymase concentration will be increased in these diseases, and by measuring the amount of chymase released in serum, it becomes possible to clarify the pathological condition of diseases involving chymase. .

[0004] For example, in Japanese Patent Laid-Open No. 11-199600, human chymase accumulated in intracellular granules in mast cells was measured, and hypertension related to human chymase or a disease related thereto and allergic property related to human chymase. A technique for analyzing the pathological condition of a disease has been reported. However, this method is a method in which only mast cells are first collected from blood and then human chymase is measured by using the enzyme activity, and the operation procedure is very complicated. Moreover, this method is to measure human chymase in mast cells, not to measure human chymase that is exposed to the living body and is directly involved in the disease, and is not suitable for diagnosing pathological conditions. Met.

Therefore, there has been a demand for a means for directly measuring the amount of human chymase exposed in serum to diagnose a disease associated with human chymase and its pathological condition, but it has been difficult to provide it in reality.

That is, serine protease enzyme inhibitors such as antichymotrypsin are contained in serum, and the presence of an endogenous enzyme inhibitor that specifically inhibits chymase has been shown. Therefore, it has been difficult to detect and measure the amount of human chymase released in serum in the enzyme-specific activity.

[0007] As a method for detecting a trace amount of components contained in serum, a sandwich ELISA (Sandwitch Enzyme-Linked Immunoso) using a monoclonal antibody is widely used.
rbent Assay) method is used. However, in order to establish the sandwich ELISA method for detecting human chymase, at least two types of antibodies with high specificity and no cross-reactivity with human chymase are required, but such antibodies are difficult to obtain. Met.

Hitherto, in order to prepare an anti-human chymase monoclonal antibody, human chymase was extracted and purified from mast cells contained in human skin tissue, and this was used as an antigen. In this method, a large amount of purified human chymase is obtained. It was not possible, and it was insufficient as an antigen. In fact, Walls et al. (J. Payhol., Vol.189, pp138-143, (199
9).) Prepared an anti-human chymase monoclonal antibody using human chymase extracted from skin tissue as an antigen, but its specificity was low.

[0009]

Accordingly, the present invention provides an anti-human chymase monoclonal antibody with high specificity,
It is an object of the present invention to provide a method that enables the measurement of human chymase in serum using this, and further to provide a test and diagnostic technique for diseases associated with human chymase.

[0010]

[Means for Solving the Problems] The present inventors previously conducted a large amount of recombinant human chymase having biological activity by a method utilizing the silkworm larva-baculovirus system with human chymase as a host, which is one of the genetic engineering techniques. Was found separately and applied for a patent (Japanese Patent Application No. 2001-1).
08186).

The present inventors have conducted extensive studies to obtain anti-human chymase monoclonal antibodies with high specificity. As a result, the degradation product obtained by enzymatic treatment of human chymase or the recombinant human chymase obtained by the above method was used as an antigen. It has been found that a highly specific anti-human chymase monoclonal antibody can be obtained by using it and that a measurement system for human chymase exposed in serum can be constructed by using these. Then, the amount of human chymase measured by the present measurement system has a correlation with diseases caused by AngII produced by human chymase and diseases caused by the action of human chymase itself. That is, it was found that the present measurement system can be used as a diagnostic agent for the above-mentioned diseases, and completed the present invention.

That is, the present invention provides an anti-human chymase monoclonal antibody against a degradation product obtained by enzymatically treating human chymase.

The present invention also provides an anti-human chymase monoclonal antibody against recombinant human chymase obtained by utilizing an insect living body of the Bombycidae or Mothidae or a cultured cell of these insects as a host.

Further, according to the present invention, an animal is immunized with a degradation product obtained by enzymatically treating human chymase, spleen cells are taken out from the animal, fused with myeloma cells, and the obtained hybridoma cells are cultured, The present invention provides a method for obtaining an anti-human chymase monoclonal antibody, which comprises collecting an antibody.

Furthermore, in the present invention, the analyte is a chelating agent,
Pretreatment is performed by incubating or heating in a solution containing an inhibitor for an enzyme that decomposes chymase and a surfactant, and then the sample is reacted with an immobilized anti-human chymase monoclonal antibody, and then a labeling substance is added. The present invention provides a method for measuring human chymase, which comprises reacting the antibody labeled with the above with an anti-human chymase antibody having a different antigen recognition site, and measuring the labeled amount of the obtained complex.

Furthermore, the present invention provides the following components (a) to
A kit for measuring human chymase containing (c) is provided. (A) Solid phase immobilized with anti-human chymase monoclonal antibody (b) Labeled with a labeling substance, anti-human chymase antibody different in antigen recognition site from the anti-human chymase monoclonal antibody of component (a) (c) Chelating agent, chymase Pretreatment Agent Containing an Inhibitor for an Enzyme that Decomposes Sulfate and a Surfactant

The present invention also provides a diagnostic agent for hypertension, a diagnostic agent for allergic disease or a diagnostic agent for cardiovascular system, which comprises the anti-human chymase monoclonal antibody.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION An anti-human chymase monoclonal antibody against a degradation product obtained by enzymatically treating human chymase of the present invention (hereinafter referred to as "anti-human chymase monoclonal antibody against degradation product") can be obtained by the following procedure. it can. That is, the degradation by a procedure of obtaining human chymase to be used as an antigen, obtaining a degradation product by enzymatic treatment of the human chymase, immunizing an animal with the degradation product and producing antibodies, producing hybridoma cells, and selecting an anti-human chymase monoclonal antibody. An anti-human chymase monoclonal antibody against the product can be obtained.

First, as human chymase used as an antigen, purified human chymase having physiological activity is preferable. As a method for obtaining the purified physiologically active human chymase, any method may be used, for example,
A method of extracting and purifying human chymase from mast cells contained in human skin tissue, a method of producing and purifying using a recombinant, or a method of infecting silkworms with recombinant baculovirus, which the present inventor has separately filed , A method for collecting and purifying body fluid (Japanese Patent Application No. 2000-108186). Furthermore, regarding the purity of this purified physiologically active human chymase, sodium dodecyl sulfate (hereinafter,
"SDS")-Polyacrylamide electrophoresis (Laem
mli, Nature 227, pp680, (1970).) (hereinafter referred to as "SDS-
(Hereinafter referred to as "PAGE"), the purity of the gel after migration is measured by Coomassie Brilliant Blue (CBB) staining method, and the purity is preferably 95% or more. Regarding the enzyme activity, the substrate AngI
In the conversion reaction from bisphenol to AngII, the Km value is 60 μM
(Kinoshita, A, J. Biol. Chem., Vol266, pp19192-7,
(1991).) Physiological activity of about (1991). Is preferable, and one having a Km value of about 67 μM is particularly preferable.

Purified physiologically active human chymase is
To obtain the recombinant baculovirus by the method, the following procedure may be performed. That is, (a) a recombinant virus having a gene encoding a protein secretion signal peptide of a host, a gene encoding an enterokinase cleavage sequence and a gene encoding human chymase is constructed, and (b) the recombinant virus is propagated. And then infecting the host or host cell, (c) after propagating this recombinant virus, obtaining inactive recombinant human chymase from the host or host cell culture supernatant, (d) purifying it and then enterokinase It is possible to obtain human chymase having a physiological activity, which is purified by the procedure of producing a human chymase by converting it into an active form.

The recombinant viruses used in the above are, for example, baculoviruses such as silkworm nuclear polyhedrosis virus (BmNPV), genes encoding host secretory protein signal peptides, genes encoding enterokinase cleavage sequences, and It has a gene encoding human chymase incorporated therein.

The gene encoding the protein secretion signal of the host (hereinafter referred to as "signal peptide gene") incorporated into the recombinant virus used above may be, for example, silkworm 30K protein when the host is silkworm. The gene which codes a signal peptide can be mentioned. This gene is described in, for example, Sakai et al. (Bioche
m. Biophys. Acta., 949; pp224-232, (1988).), and can be obtained based on this.

Further, a gene encoding human chymase (hereinafter referred to as "human chymase gene") has been reported by, for example, Takai et al. (FEBS. Let., 412, pp86-90, (1997).). It can be obtained by performing RT-PCR based on the literature.

Furthermore, a gene encoding an enterokinase cleavage sequence can be added to the human chymase gene by providing an enterokinase cleavage site in the primer sequence or by using a cloning vector such as pBluBacHis2B (manufactured by INVITROGEN). is there.

Each of these genes is preferably inserted downstream of the viral polyhedrin promoter in the order of the signal peptide gene, the gene encoding the enterokinase cleavage sequence and the human chymase gene.

The binding of each of these genes is carried out by using a suitable primer, if necessary, after obtaining the above-mentioned gene by a conventional method.
After digestion with a restriction enzyme, ligation may be performed using a ligation kit. Specifically, cDNA amplified by PCR using a primer having an appropriate restriction enzyme site
This can be carried out by digesting the fragment with a restriction enzyme and then mixing the transfer vector previously digested with the same restriction enzyme with the ligation reagent.

In addition, amplification of the human chymase gene, acquisition thereof, or integration into the virus can be carried out by conventional methods. For example, for amplification and acquisition of the human chymase gene, PCR is performed using appropriate primers.
It may be carried out by a reaction, and the integration into the virus is performed by Maeda et al. (Annu. Rev. Entomol., 34; pp351-372).
(1989).).

The recombinant virus thus obtained is then allowed to infect a host and propagated. The host used may be the host of the virus itself or a cultured cell derived from the host. Specifically, as the host used for the production of the recombinant human chymase of the present invention, if the recombinant virus is based on BmNPV, silkworm worms, pupa or silkworm cultured cells are used. .

As a method for infecting a host or a cultured cell of the host with the recombinant virus and a method for proliferating after infection, known methods can be used. For example, when silkworms are used as the host, ingestion of the recombinant virus into them can be carried out by oral infection or infection by injection.

As a method for separating and collecting the inactive recombinant human chymase produced by the above-described infection or proliferation method from the host or the cultured cells of the host, known methods can be used. For example, when a silkworm worm is used as a host, it can be performed as follows. That is, first, a body fluid is collected from a silkworm that has been infected with a recombinant virus and bred, and this is purified.

Purification of inactive recombinant human chymase from the silkworm worm body fluid is preferably carried out by a cation exchange column,
It is then carried out by subjecting it to column chromatography in the order of the heparin column. Among these, it is preferable to perform the cation exchange column chromatography at a relatively high pH (for example, around pH 7.8) because it is easy to separate from other miscellaneous proteins in the silkworm body fluid. In addition,
There is a method of using an anion exchange column, a gel filtration column, or a heparin column as the primary column. However, in this method, the amount of other miscellaneous proteins mixed in is large,
Isolation of inactive recombinant human chymase is difficult and is not a preferred method.

Since the inactive recombinant human chymase thus obtained has an enterokinase cleavage sequence added thereto, it is activated. This process
It is carried out by using enterokinase, for example, by incubating at a temperature of about 25 ° C. for 6 hours or more.

By performing a separation treatment with a column after the above treatment, the contaminating enterokinase-cleaving peptide and enterokinase can be removed, and only the desired recombinant human chymase having physiological activity can be obtained. Heparin column chromatography is preferably used to remove the cleaved enterokinase cleaved peptide and enterokinase and to isolate the bioactive recombinant human chymase.

The thus obtained physiologically active recombinant human chymase has almost the same physical properties as the natural human chymase, but is clearly different in that it is highly stable. For example, general culture conditions of animal cultured cells, that is, MEM medium containing 5% FBS (minimum esse)
ntial medium (manufactured by Sigma) and incubated at 37 ° C. in a constant temperature bath, natural human chymase immediately lost chymase activity, but this recombinant human chymase retained chymase activity for several days.

On the other hand, to prepare an anti-human chymase monoclonal antibody from purified physiologically active human chymase, human chymase may be once treated with an enzyme to give a degradation product, which may be used as an antigen.

Enzymatic treatment of human chymase is carried out by treating human chymase with trypsin, chymotrypsin, thermolysin, V
8 Treatment with an enzyme such as protease or lysyl endopeptidase is performed. For example, when lysyl endopeptidase is used, this enzyme treatment may be performed in a PBS solution having an enzyme concentration of 2 μg / ml to 3 μg / ml at a temperature of 28 ° C. to 32 ° C. The amount of lysyl endopeptidase for human chymase at this time is about 1/400 of that for human chymase, and may be about 2 μg to 3 μg. Whether or not a degradation product suitable for the production of the human chymase monoclonal antibody of the present invention was obtained can be determined by SDS-PAGE and then staining the gel with a silver staining method.

The immunization of animals with the degradation products obtained by the enzyme treatment of the above human chymase is carried out by, for example, mixing this with a complete adjuvant or an incomplete adjuvant according to a conventional method, and repeating the same until mice and rabbits have increased their immunopotency. Etc. by intraperitoneal injection of the animal. Then, spleen cells are taken out from the animal, and further fused with myeloma cells such as mouse by an appropriate means, for example, in the presence of polyethylene glycol or by means of electroporation or the like, to produce an anti-human chymase monoclonal antibody hybridoma cell. To get Then, by culturing the hybridoma cells in an appropriate medium, an anti-human chymase monoclonal antibody against the degradation product can be obtained. Needless to say, the method for producing the anti-human chymase monoclonal antibody against the degradation product is not particularly limited to this method.

From the anti-human chymase monoclonal antibodies produced by the hybridoma cells obtained as described above, first, an antibody with high specificity for human chymase is selected, and then the anti-human chymase having different antigen recognition sites is selected by the following screening method. Monoclonal antibodies were selected.

Screening for anti-human chymase monoclonal antibodies having different antigen recognition sites was carried out as follows. That is, for example, after lysyl endopeptidase-treated degradation product of the recombinant human chymase having physiological activity produced and purified as described above is electrophoresed by SDS-PAGE, the antigen protein is converted from gel to polyvinylidene fluoride (hereinafter referred to as “PVDF”) by a blotting device. ) Transfer to the membrane and make a PVDF membrane with the adsorbed decomposition products.
Anti-human chymase monoclonal antibody produced from the hybridoma cells obtained as above as a secondary antibody, 2
Using a peroxidase-labeled anti-mouse IgG rabbit antibody as the next antibody, immunoblotting was performed to confirm the antigen recognition site, and hybridoma cells producing anti-human chymase monoclonal antibodies having different antigen specificities were selected. By culturing these hybridoma cells, anti-human chymase monoclonal antibody can be obtained. Since the anti-human chymase monoclonal antibody thus obtained has high specificity, it can be used as both a primary antibody and a secondary antibody in the Elisa method, for example.

The representative 2D11G10D of the hybridoma cells producing the anti-human chymase monoclonal antibody obtained as described above was designated as FERM P-18700 on February 8, 2002, National Institute of Advanced Industrial Science and Technology Depositary Center (Zip Code 305-8
566 Deposited at 1-chome, 1-chome, 1-chome, Tsukuba-shi, Ibaraki, Japan, 6).

In addition, an anti-human chymase monoclonal antibody against recombinant human chymase obtained by utilizing an insect living organism of the Bombycidae or Mothidae or a cultured cell of these insects as a host (hereinafter referred to as "anti-human chymase monoclonal against recombinant human chymase"). In the procedure for producing an anti-human chymase monoclonal antibody against the above degradation product, a silkworm is infected with the above-mentioned recombinant baculovirus to the antigen used in the procedure, and a body fluid is collected and purified (Japanese Patent Application No. 2001). -108186)
It can be obtained by substituting the recombinant human chymase obtained by the above, and then performing the procedure.

The anti-human chymase monoclonal antibody against the degradation product of the present invention and the anti-human chymase monoclonal antibody against recombinant human chymase (hereinafter, referred to as "anti-human chymase monoclonal antibody of the present invention") are immunological studies of human chymase present in serum or the like. It can be used for dynamic measurement. As the immunological measurement method, a radioisotope immunoassay method (RIA method), an ELISA method, a fluorescent antibody method, a plaque method, a spot method, an agglutination method, an Ouchterlony method, etc.,
Although various methods used in general immunochemical assay methods can be used, the Eliza method is preferred in terms of sensitivity, simplicity, and the like. Also in the ELISA method, the sandwich-ELISA method in which a primary antibody that is immobilized on a solid phase and captures a substance to be detected and a secondary antibody that is labeled and shows the amount of the substance to be detected is preferable.

In the above sandwich-Eliza method,
As the secondary antibody, in addition to the anti-human chymase monoclonal antibody of the present invention, a polyclonal antibody against human chymase can be used. In order to prepare such an anti-human chymase polyclonal antibody, for example, the human chymase having the physiological activity produced and purified as described above is directly used as an antigen, and this is mixed with complete adjuvant or incomplete adjuvant according to a conventional method. It can be obtained by repeatedly injecting this into animals such as guinea pigs and rabbits until the immunopotency of the anti-human chymase polyclonal antibody in serum increases. And
Three days after the final immunization, whole blood is collected, and from this, blood clots are precipitated and separated by centrifugation, whereby serum containing anti-human chymase polyclonal antibody can be obtained.

By using the above-described anti-human chymase monoclonal antibody or anti-human chymase polyclonal antibody of the present invention, a system for measuring human chymase in various specimens can be constructed.

However, it may be insufficient when actually measuring human chymase in a biological sample such as serum. That is, in biological samples, the presence of serine protease enzyme inhibitors such as antichymotrypsin or endogenous enzyme inhibitors that specifically inhibit human chymase is known, and human chymase released in serum is complexed with these. It is highly possible that the body is formed, and it may mask the antigen-determining site of the anti-human chymase monoclonal antibody of the present invention, and it is difficult to accurately detect and measure human chymase in a biological sample. it is conceivable that. Further, since the human chymase monoclonal antibody of the present invention also recognizes an antigenic determinant site that recognizes human chymase, since it is possible that it is inside human chymase, it is possible to accurately detect and measure human chymase without treatment to expose the antigenic determinant site. Is also considered difficult.

Therefore, prior to the measurement of human chymase, it is preferable to pretreat a sample containing human chymase such as serum to release human chymase. Specifically, it is preferable to add a solution containing a chelating agent, an inhibitor for an enzyme that decomposes chymase, and a surfactant to a sample for which human chymase is to be measured, and to heat or heat the solution therein. The chelating agent used in this solution is ethylenediaminetetraacetic acid (hereinafter referred to as "EDT
Abbreviated as “A”), ethylene glycol-bis- (β-aminoethyl ether) -N, N, N ′, N′-4 acetic acid (hereinafter,
(Abbreviated as “EGTA”) and the like, and as an inhibitor for an enzyme that decomposes chymase, for example, phenylmethylsulfonyl fluoride (hereinafter, referred to as “PMSF”) that is an inhibitor for protease in serum is used. As the surfactant, SDS, Tween 20 or the like is used. The concentration of the chelating agent in this solution is about 0.1 mM to 5 mM, the concentration of the inhibitor for the enzyme that decomposes chymase is about 0.1 mM to 5 mM, and the concentration of the surfactant is about 0.01% to 5%. preferable. Further, this solution is, for example, 50 mM Tris-H.
Cl (pH 8.0), T consisting of 150 mM NaCl
It may be diluted with a BS solution or the like.

The heat retention or heat treatment in the above solution may be carried out within the range of 25 ° C. to 100 ° C., and 80 ° C.
To 100 ° C. is more preferable.

The specimen pretreated as described above is then treated with the anti-human chymase monoclonal antibody of the present invention (hereinafter referred to as "1
The secondary antibody "is reacted with the immobilized solid phase, and then the anti-human chymase antibody labeled with a labeling substance and having an antigen recognition site different from that of the anti-human chymase monoclonal antibody (hereinafter referred to as" secondary antibody ") is reacted. Then, human chymase can be measured from the labeled amount of the obtained complex.

In the above sandwich-Eliza method, 1
The support (solid phase) used for immobilizing the next antibody is not particularly limited as long as it can immobilize the above antibody in a sufficient amount, and a plate made of a substance usually used as a support is used as a raw material. , Membrane, beads, gel, etc. can be used. However, in order to quantitatively measure the amount of human chymase in serum, it is preferable that a dilution series of a serum sample be prepared and measured at one time, and it is particularly preferable to use a 96-well immuno plate as a support. In addition, as a method for immobilizing on a support, covalent bond,
Examples thereof include a method using a non-covalent bond and a cross-linking agent, but the method is not particularly limited as long as a sufficient amount of the primary antibody can be immobilized on the solid phase.

In the sandwich ELISA method, the secondary antibody for detecting human chymase bound to the immobilized primary antibody may be an anti-human chymase polyclonal antibody as long as it has an antigen recognition site different from that of the primary antibody. Although either an anti-human chymase monoclonal antibody may be used, the anti-human chymase monoclonal antibody of the present invention having a different antigen recognition site from the primary antibody prepared above is particularly preferable.

The labeling substance used for labeling these secondary antibodies is preferably one that can be detected with high sensitivity even in a small amount. Examples of such labeling substances include alkaline phosphatase, peroxidase, β
-Enzymes such as galactosidase, radioisotopes, GF
Fluorescent substances such as P and fluorescein, bioluminescent enzymes such as luciferase, and the like. As a method of binding the labeling substance to the binding protein, a known binding method to the protein may be used. Of course, not only a method of directly binding the above-mentioned labeling substance to the antibody, but also an indirect binding method such as utilizing an antibody to which biotin or the like is bound and binding the labeling substance bound to streptavidin or the like to it There is no problem even if it is used.

Further, in the sandwich ELISA method, the amount of human chymase is detected and measured by measuring the amount of labeled binding protein. In the measuring method of the present invention, the amount of human chymase is measured by measuring the labeling substance of the labeled binding protein, and the measuring device used for these is a spectrophotometer, a microplate reader, a liquid scintillation counter, a fluorescence spectrophotometer. It can be appropriately selected and used according to the labeling substance such as and luminometer.

In order to easily carry out the method for measuring human chymase of the present invention, for example, a human chymase measuring kit containing the following components (a) to (c) can be used. (A) Solid phase immobilized with anti-human chymase monoclonal antibody (b) Labeled with a labeling substance, anti-human chymase antibody different in antigen recognition site from the anti-human chymase monoclonal antibody of component (a) (c) Chelating agent, chymase Pretreatment Agent Containing an Inhibitor for an Enzyme that Decomposes Sulfate and a Surfactant

According to the method of the present invention described above, the amount of human chymase contained in a biological sample such as serum can be measured with high accuracy. And, AngII produced by human chymase is recompressed after percutaneous coronary angioplasty, vascular lesion such as bypass graph and intimal cavity thickness after execution, hypertension, cardiac hypertrophy, myocardial infarction, arteriosclerosis, diabetes. Heart and cardiovascular diseases such as non-diabetic renal disease and diseases such as solid tumors, and chymase itself also has allergic properties such as rheumatoid arthritis, keloids, psoriasis, and atopic dermatitis. Involved in the disease,
The test agent containing the anti-human chymase monoclonal antibody of the present invention can be used as a diagnostic agent for diagnosing the pathological condition of the above-mentioned diseases, examination of prognosis of treatment and the like. Further, it can also be used as a clinical test reagent for clinical evaluation of chymase inhibitors.

[0055]

In the present invention, as an antigen for obtaining an anti-human chymase monoclonal antibody, not human chymase but an enzyme-treated one thereof is used, and the specificity of recognizing an antigen recognition site which has never been utilized hitherto is used. High anti-human chymase monoclonal antibody was obtained.

Further, in the present invention, as an antigen for obtaining an anti-human chymase monoclonal antibody, not a human-derived human chymase, but an insect living organism of the Bombycidae or Mothidae or a cultured cell of these insects is used as a host. By using the recombinant human chymase, an anti-human chymase monoclonal antibody having higher specificity than conventional ones was obtained.

The specimen is also pretreated prior to the immunological measurement to expose the above-mentioned antigen recognition site in human chymase to enhance the accuracy of measurement of human chymase in serum.

Therefore, the present invention provides clinical studies such as diagnosis of human chymase and various diseases caused by AngII produced by the chymase, medical examination for the purpose of prevention, prognostic examination for treatment and clinical evaluation of chymase inhibitors. It can be used in the development of test reagents.

[0059]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 Construction of virus expressing inactive recombinant human chymase (hereinafter abbreviated as EK-chymase) in which enterokinase cleavage sequence (DDDDDK) was added to N-terminal: (1) Cloning of human chymase cDNA (a) Obtaining human chymase cDNA Human chymase cDNA is a cDNA synthesis kit (Gibco
It was performed by the RT-PCR method using BML). First, using 4 μg of total RNA extracted from human blood vessels,
Based on the nucleotide sequence of human chymase reported by Taka et al. (Takai et al. (FEBS.Let., 412, pp86-90, (1997)).), The following sense and antisense primers were designed. The human chymase cDNA was amplified by carrying out RT-PCR reaction for 40 times at 94 ° C. for 1 minute, 63 ° C. for 1 minute, and 72 ° C. for 1 minute.

Sequence of sense primer: 5'-TTGCTCAGGCAGCCTCTCTG-
Sequence of 3'antisense primer: 5'-TTCAGGCTGGCTCAGGATCC-
3 '

(B) Construction of a cloning vector in which an enterokinase cleavage site is added to the N-terminal of human chymase: First, using the human chymase cDNA obtained above as a template, the nucleotide sequence of human chymase previously reported (Takai et al. (FEBS . Let., 412, pp86-90, (1997)).) For designing sense (His / EK-CHY) and antisense primer (CHY-salI) having the following enterokinase cleavage sites. PCR reaction was carried out by a conventional method using the EK-chymase cDNA.
I got A. The obtained EK-chymase cDNA was treated with restriction enzymes SphI and SalI, and then subcloned between SphI-SalI of cloning vector pQE31 (manufactured by QIAGEN) to construct pQE / EK-CHY.

Sense primer (His / EK-CH
Y) sequence: 5'-CCGCATGCCGATGGACGATGACA
Sequence of AAATCATCGGGGGCACAGAATG-3 'antisense primer (CHY-SalI): 5'-GAGCAGCTGAATTTAAACGGACG
ACGTCCTAG-3 '

(2) Construction of transfer vector having secretory signal sequence of silkworm-derived secretory protein 30K protein: Nucleotide sequence of secretory signal of secretory protein "30K protein" derived from silkworm (Sakai et al., Bi
ochim. Biophys. Acta 949, 224-232 (1988).),
Oligonucleotides for the sense and antisense strands were synthesized.

These were treated with 9 mM in the presence of 50 mM NaCl.
By annealing at 55 ° C. for 55 minutes, at 55 ° C. for 15 minutes, and at 37 ° C. for 15 minutes, a signal oligo DNA having a secretory signal sequence of 30K protein was obtained. A transfer vector (pYNGsig) having a 30K protein secretion signal sequence was constructed by inserting this signal oligo DNA between BglII-SacI of pYNG transfer vector (Katakura industry) by a ligation reaction (Ligation Kit, TaKaRa).

(3) Construction of transfer vector into which sig-EK-chymase gene was introduced: As shown in FIG. 1, pYNGsig transfer vector was transformed with EK-.
In order to introduce the chymase cDNA, it was once subcloned into a pTZ8 cassette vector (manufactured by Katakura Industries) and a restriction enzyme site was newly added. That is, pQE / E
K-CHY was digested with restriction enzymes SphI and SalI to excise an EK-chymase cDNA fragment, which was digested with pT
It was inserted into the SphI-SalI restriction enzyme site of Z8 (pTZ8EKChym).

Next, pTZ8EKChym was digested with a restriction enzyme XbaI, and then a blunting kit (Bl
Unting Kit (TaKaRa) was used to make blunt ends, and further digested with the restriction enzyme EcoRI to obtain EK-chymase c.
The DNA was cut out. This is the Sma of pYNGsig
By inserting it between I-EcoRI, a 30K protein secretion signal sequence, E, is provided downstream of the polyhedrin promoter.
A transfer vector (pYNGsigEKChym) into which genes were introduced in the order of K-chymase sequence was obtained (FIG. 1). The sequence between the 30K protein secretion signal and EK-chymase of the transfer vector is shown in FIG.

(4) Preparation of sig-EK-chymase gene recombinant baculovirus Recombinant baculovirus was prepared by Maeda et al.
al., Annu. Rev. Entomol., 34; pp351-372 (1989).)
The method was modified. That is, first pYNGsi
The gEKChym plasmid DNA was purified by a plasmid purification kit (QIAGEN). Next, linearized baculovirus (CPd strain: Japanese Patent No. 3,090,586, US Pat. No. 5,753,2)
20) DNA and purified pYNGsigEKChy
mPlasmid DNA was mixed, and BmN cells (Maeda et al., Ann) were treated with lipofectin reagent (manufactured by Gibco BML).
u. Rev. Entomol., 34; pp351-372 (1989).).

The recombinant baculovirus was screened by the limiting dilution method (Maeda et a
l., Annu. Rev. Entomol., 34; pp351-372 (1989).) to select viruses that do not form polyhedra. Next, the virus-infected cells were collected and the EK-chymase cDNA was used.
Using the A probe, Southern blot analysis and Northern blot analysis were performed to select and clone a recombinant virus in which expression of EK-chymase mRNA was found and in which the EK-chymase gene was introduced into the viral genome ( CPdsigEKChym virus).

Example 2 Production and purification of recombinant human chymase in silkworms: (1) Production of EK-chymase in silkworm larvae CPdsigEKChym virus was inoculated into silkworms on the 5th day on the 5th day of virus infection. Body fluid was collected. The collected body fluid was centrifuged at 25,000 rpm for 60 minutes to collect the supernatant and remove contaminated tissue pieces and the like. A 3-fold amount of 50 mM sodium phosphate (buffer A: pH 7.8) was added to the obtained supernatant to prepare a body fluid containing EK-chymase.

(2) Purification of recombinant human chymase expressed in silkworm body fluid: (a) Purification of EK-chymase by column chromatography Sp Toyopearl cation exchange column (manufactured by Tosoh) was used as buffer A (equilibration buffer). After equilibrating against
Silkworm body fluid containing EK-chymase was added and adsorbed. Then, after washing with an equilibration buffer containing 0.2 M NaCl, EK-chymase was eluted by increasing the salt concentration to 0.5 M NaCl (in FIG. 3,
(A)). The fraction containing EK-chymase was 50 mM Tr.
The final concentration of NaCl was adjusted to 250 mM by adding an equal amount of is-HCl (pH 8.0) (buffer B). Subsequently, a heparin affinity column (HiTrap Heparin, Pharmacia) was added to buffer B.
After equilibration with respect to, the fraction containing the recombinant human EK-chymase recovered from the cation exchange column was added and adsorbed. 0.4 after washing with equilibration buffer first
Buffer B containing M NaCl (washing buffer)
Washed again with. Elution of recombinant human EK-chymase
The concentration of salt in buffer B was applied by applying a linear gradient of NaCl of 0.4 to 1.0 M, and recombinant human EK-chymase was eluted under the condition of salt concentration of 0.5 M NaCl (FIG. 3). (B)).

(B) Maturation treatment of recombinant human EK-chymase 45 units of enterokinase (Funakoshi) was added per 1 μg of the recombinant human EK-chymase obtained in (a) above, and the mixture was incubated at 25 ° C. for 6 hours or more. By doing so, the enterokinase cleaving peptide attached to the N-terminus of the recombinant human EK-chymase was removed, and a mature recombinant human chymase having enzymatic activity was obtained.

(C) Purification of matured recombinant human chymase To remove the enterokinase cleaving peptide and enterokinase from the matured recombinant human chymase having the enzymatic activity obtained in (b) above, affinity with a heparin column was used again. Purification is carried out in the same manner as in (a), 0.4-1.0M
Recombinant human chymase matured by a linear gradient of NaCl was eluted and isolated (in FIG. 3,
(C)). All the steps related to column operation were performed at 4 ° C. The isolated recombinant human chymase is SDS-
The electrophoresis was performed by PAGE, and the purity was measured by the CBB staining method. As a result, the purity of recombinant human chymase was 95%. The recombinant human chymase thus obtained had a Km value of 67 μM and showed almost the same physical properties as the natural human chymase, but was clearly different in that it had high stability.

Example 3 Preparation of Anti-Human Chymase Monoclonal Antibody Against Degradation Products: (1) Preparation of Antigen The matured recombinant human chymase prepared in Example 2 above was lysyl endopeptidase (manufactured by Wako Pure Chemical Industries). Was treated with an enzyme. The amount of lysyl endopeptidase used for human chymase is 1/400 that of human chymase, and lysyl endopeptidase is
Used at a concentration of 2.5 μg / ml in BS. The treatment was limited decomposition under the conditions of 30 ° C. and 6 hours. In the following examples, the degradation product obtained as described above (hereinafter sometimes simply referred to as "degradation product") was used as an antigen.

(2) Preparation of hybridoma cells The degradation products prepared in (1) above were mixed with complete adjuvant or incomplete adjuvant (both manufactured by Difco), and 8 weeks old female mice (BALB) were prepared.
/ C; purchased from Saitama Experimental Animal) was intraperitoneally injected at a dose of 50 μg per human chymase.
This operation was repeated several times, and on the third day after the final immunization, the spleen was extracted from the mouse whose antibody titer had increased. Spleen cells taken out from the spleen were mixed with mouse myeloma cells at a ratio of 5: 1, and 50% polyethylene glycol 1500 was added.
(Manufactured by Roche Diagnostics) was mixed as a fusion promoter and cell fusion was performed to prepare hybridoma cells. After cell fusion, the hybridoma cells were 5 × 10 ⁵
HA containing 10% FBS to achieve cells / ml
Suspend in T medium, and in a 96-well microtiter plate (Falcon), 1 × 10 ⁵ cells / well
It was dispensed so that it would be 1 s.

Example 4 Preparation of Anti-Human Chymase Monoclonal Antibody Against Recombinant Human Chymase: The matured recombinant human chymase prepared in Example 2 above was used as an antigen as it was, and hybridoma cells were prepared in the same manner as in Example 3 above. Created.

Example 5 Screening of Hybridoma Cells Producing Anti-Human Chymase Monoclonal Antibodies with Different Antigen Specificity: SDS was applied to the degradation products prepared in (1) of Example 3 above.
-Electrophoresis was performed by PAGE. After the electrophoresis was completed, the protein was transferred from the gel to a PVDF membrane (manufactured by Advantech) using a blotting device (manufactured by Atto). PVDF membrane with this protein adsorbed on it is 2% skim milk (Snow Brand Milk Products), 0.05% Tween 20, 50m
M Tris-HCl solution (pH 8.0) and 150
Anti-human chymase monoclonal antibody produced from the hybridoma cells obtained in Example 3 or Example 4 as a primary antibody after blocking treatment with a blocking solution containing mM NaCl solution (hereinafter, simply referred to as “blocking solution”) Immunoblotting was performed using an antibody and a peroxidase-labeled anti-mouse IgG rabbit antibody (manufactured by Wako Pure Chemical Industries, Ltd.) as the secondary antibody. The signal was detected by using 3,3′-diaminobenzidine (DAB: manufactured by Wako Pure Chemical Industries, Ltd.) to develop a color on the PVDF film.

As a result, among the hybridoma cells obtained in Examples 3 and 4, some hybridoma cells producing anti-human chymase monoclonal antibody having different antigen recognition sites were obtained. For example, an anti-human chymase monoclonal antibody produced by the hybridoma cell of clone number 6D obtained in Example 4 (hereinafter referred to as "HuC
hym (6D) Mab ”) and the anti-human chymase monoclonal antibody produced by the hybridoma cells of clone number 2D11G10D obtained in Example 3 (hereinafter,
"HuChym (2D11G10D) Mab" is abbreviated)
Regarding, the antigen recognition sites were compared, and it was confirmed that there were bands in which the antigen recognition sites did not match (lanes 1 and 2 in FIG. 4). A conventional anti-human chymase monoclonal antibody (manufactured by CHEMICON INTERNATIONAL INC.) Could not recognize the above antigen (lane 3 in Fig. 4).

Example 6 Preparation and purification of anti-human chymase monoclonal antibody:
To produce large amounts of monoclonal antibodies, pristane (2,6,10,14-tetramethylpentadecane) was added to 0.1%.
5 ml was intraperitoneally administered and raised for 2 to 4 weeks, 8 to 10
2-10 × 10 ⁶ cells / hour of the anti-human chymase monoclonal antibody-producing hybridoma cells obtained in Example 3 or Example 4 were added to week-old female mice (BALB / c).
It was intraperitoneally administered in a rat. Hybridomas develop ascites cancer in 10 to 21 days, and ascites was collected from these mice.
Centrifugation was performed at 000 rpm for 5 minutes to remove solids, and the supernatant was passed through a Protein A (Protein A) column (manufactured by Amersham Pharmacia Biotech), and the adsorbed fractions were collected to obtain a purified monoclonal antibody IgG fraction.
The IgG concentration is based on Coomassie Protei
n Assay Reagent (manufactured by PIERCE).

Example 7 Measurement of Human Chymase Level in Serum by Sandwich Eliza Method: (1) Sample-purified human blood vessel-derived human chymase and serum collected from patients with renal hypertension and healthy subjects (both Osaka Medical University, Faculty of Pharmaceutical Sciences Miyazaki (Provided by the professor) was used as a sample, which was stored at −80 ° C. until used for measurement.

(2) Preparation of antibody-immobilized plate First, HuChym (6D) Mab was adjusted to 2 μg / ml with 50 mM Tris-HCl (pH 8.0),
It was diluted with 150 mM NaCl (hereinafter, abbreviated as "TBS"). This diluted solution is used for Immuno Plate 430341.
Dispense 100 μl each into each well (made by Nalje Nunc International), and use HuChym (6D) Ma
b was bound to the plate. Then, discard the solution containing unadsorbed antibody in the wells and add 100 μl of blocking solution to each well.
The plate was blocked by aliquoting each of them and incubating at 37 ° C for 1 hour. Then, the blocking solution was discarded, and TBS containing 0.05% Tween 20 (hereinafter, abbreviated as "T-TBS") was added to each plate in 3 wells.
It was dispensed by 00 μl each, and the plate was washed 4 times to prepare an antigen-immobilized (antigen capture) plate.

(3) Preparation of Detection Antibody HuChym (2D11G10D) Mab was prepared by using ECL protein biotinylation system (Amersham Pharmacia Biotech) to prepare biotin and HuChym.
HuChym (2D11G10D) Mab was mixed and incubated at room temperature for 1 hour.
10D) Mab and biotin were cross-linked to prepare biotin-labeled HuChym (2D11G10D) Mab.

(4) Release treatment of human chymase in sample Purified human blood vessel-derived human chymase and serum were 5 m
Diluted 2-fold with TBS containing M EDTA, 5 mM EGTA, 1 mM PMSF and 0.1% SDS,
The sample was subjected to boiling treatment at 100 ° C. for 3 minutes to give a sandwich / elizae sample.

(5) Measurement of the amount of human chymase in serum The blocked antibody-immobilized plate prepared in (2) above was subjected to 5-fold serial dilution with the blocking solution of the serum liberated in (4) above, and 37 Primary antigen-antibody reaction was performed by incubating at 60 ° C. for 60 minutes. After the reaction, the reaction solution was discarded from the plate, 300 μl of T-TBS was further dispensed into each well of the plate, and the plate was washed 4 times. Then, biotin-labeled HuCh
ym (2D11G10D) Mab was diluted with a blocking solution, 100 μl was added to each well, and the mixture was incubated at 37 ° C. for 60 minutes to carry out a secondary antigen-antibody reaction. After the reaction, the reaction solution was discarded from the plate, 300 μl of T-TBS was further dispensed into each well, and the plate was washed 4 times. 1) Horseradish peroxidase-labeled streptavidin (Amersham Pharmacia Biotech) diluted to 5,000 cultures with T-TBS was added to the antibody-immobilized plate after the secondary antigen-antibody reaction.
Then, 00 μl was added, and the mixture was incubated at 37 ° C. for 30 minutes to bind biotin with horseradish peroxidase-labeled streptavidin. The well solution is discarded, the well is washed, and 100 μl of TMB reagent (manufactured by KPL) is used.
In addition, color was developed by incubating at room temperature for 30 minutes. After 30 minutes, add 0.5 N H ₂ SO ₄ to 1
The reaction was stopped by adding 00 μl, and the absorbance was measured by a plate reader (BioRad) at 450-
It was measured at a wavelength of 650 nm.

As a result, first, the purified human blood vessel-derived human chymase was measured for untreated and treated, and it was 10 μg / ml to 0.1 only when pretreated.
It was found that human chymase can be detected in the range of μg / ml (FIG. 5). Next, when serum samples of renal hypertension patients and healthy subjects were pretreated and the amount of chymase was measured, it was possible to detect chymase in the range where serum was diluted to 1250 times. Furthermore, when the renal hypertensive patient and the healthy person were compared, the chymase amount in the serum of the renal hypertensive patient was increased from 8 times to 11.3 times that of the healthy person. From this result, it was possible to determine that certain hypertension was a disease associated with chymase.

[0086]

By using the anti-human chymase monoclonal antibody of the present invention for measuring human chymase, human chymase can be measured more easily than before. In the above measurement, human chymase in the sample can be measured with high sensitivity by pretreating human chymase in the sample.

Therefore, the present invention relates to diseases caused by human chymase, specifically, vascular lesions (restenosis after PTCA, intimal thickening after bypass grafting),
Hypertension, cardiac hypertrophy, myocardial infarction, arteriosclerosis, cardiovascular diseases such as diabetic and non-diabetic renal diseases, solid tumors and allergic diseases associated with chymase (eg, rheumatoid arthritis, keloids, psoriasis, atopy) It is used in the development of clinical test reagents for the diagnosis of the pathological condition of the most well-studied diseases such as dermatitis), screening for the purpose of prevention, prognosis and progress of treatment, and clinical evaluation of chymase inhibitors.

[0088]

[Sequence list] SEQUENCE LISTING <110> Katakura Industries Co., Ltd. <120> MONOCLONAL ANTIBODY FOR HUMAN CHYMASE AND ITS       UTILIZATION <130> 0110151 <140> <141> <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 789 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (1) .. (786) <223> EK-Chymase <220> <221> sig_peptide <222> (1) .. (57) <223> signal sequence originated in 30K-protein from       silkworm hemolymph <220> <221> unsure <222> (58) .. (93) <223> linker gene sequence <220> <221> gene <222> (94) .. (108) <223> enterokinase recognition site <220> <221> mat_peptide <222> (109) .. (786) <223> mature-Chymase <300> <302> Cloning of the gene and cDNA for human heart chymase <303> J. Biol. Chem. <304> 266 <306> 17173-17179 <307> 1991 <400> 1 atg aga ctg act ttg ttt gcc ttc gtc ctc gcc gtg tgt gcg ctg gct 48 Met Arg Leu Thr Leu Phe Ala Phe Val Leu Ala Val Cys Ala Leu Ala     -35 -30 -25 tct aac gcc aga gct ccc cta gat atc cat gca agc ttg cat gcc gat 96 Ser Asn Ala Arg Ala Pro Leu Asp Ile His Ala Ser Leu His Ala Asp -20 -15 -10 -5 gac gat gac aaa atc atc ggg ggc aca gaa tgc aag cca cat tcc cgc 144 Asp Asp Asp Lys Ile Ile Gly Gly Thr Glu Cys Lys Pro His Ser Arg              -1 1 5 10 ccc tac atg gcc tac ctg gaa att gta act tcc aac ggt ccc tca aaa 192 Pro Tyr Met Ala Tyr Leu Glu Ile Val Thr Ser Asn Gly Pro Ser Lys          15 20 25 ttt tgt ggt ggt ttc ctt ata aga cgg aac ttt gtg ctg acg gct gct 240 Phe Cys Gly Gly Phe Leu Ile Arg Arg Asn Phe Val Leu Thr Ala Ala      30 35 40 cat tgt gca gga agg tct ata aca gtc acc ctt gga gcc cat aac ata 288 His Cys Ala Gly Arg Ser Ile Thr Val Thr Leu Gly Ala His Asn Ile  45 50 55 60 aca gag gaa gaa gac aca tgg cag aag ctt gag gtt ata aag caa ttc 336 Thr Glu Glu Glu Asp Thr Trp Gln Lys Leu Glu Val Ile Lys Gln Phe                  65 70 75 cgt cat cca aaa tat aac act tct act ctt cac cac gat atc atg tta 384 Arg His Pro Lys Tyr Asn Thr Ser Thr Leu His His Asp Ile Met Leu              80 85 90 cta aag ttg aag gag aaa gcc agc ctg acc ctg gct gtg ggg aca ctc 432 Leu Lys Leu Lys Glu Lys Ala Ser Leu Thr Leu Ala Val Gly Thr Leu          95 100 105 ccc ttc cca tca caa ttc aac ttt gtc cca cct ggg aga atg tgc cgg 480 Pro Phe Pro Ser Gln Phe Asn Phe Val Pro Pro Gly Arg Met Cys Arg     110 115 120 gtg gct ggc tgg gga aga aca ggt gtg ttg aag ccg ggc tca gac act 528 Val Ala Gly Trp Gly Arg Thr Gly Val Leu Lys Pro Gly Ser Asp Thr 125 130 135 140 ctg caa gag gtg aag ctg aga ctc atg gat ccc cag gcc tgc agc cac 576 Leu Gln Glu Val Lys Leu Arg Leu Met Asp Pro Gln Ala Cys Ser His                 145 150 155 ttc aga gac ttt gac cac aat ctt cag ctg tgt gtg ggc aat ccc agg 624 Phe Arg Asp Phe Asp His Asn Leu Gln Leu Cys Val Gly Asn Pro Arg             160 165 170 aag aca aaa tct gca ttt aag gga gac tct ggg ggc cct ctt ctg tgt 672 Lys Thr Lys Ser Ala Phe Lys Gly Asp Ser Gly Gly Pro Leu Leu Cys         175 180 185 gct ggg gtg gcc cag ggc atc gta tcc tat gga cgg tcg gat gca aag 720 Ala Gly Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Ser Asp Ala Lys     190 195 200 ccc cct gct gtc ttc acc cga atc tcc cat tac cgg ccc tgg atc aac 768 Pro Pro Ala Val Phe Thr Arg Ile Ser His Tyr Arg Pro Trp Ile Asn 205 210 215 220 cag atc ctg cag gca aat taa 789 Gln Ile Leu Gln Ala Asn                 225 <210> 2 <211> 262 <212> PRT <213> Artificial Sequence <400> 2 Met Arg Leu Thr Leu Phe Ala Phe Val Leu Ala Val Cys Ala Leu Ala     -35 -30 -25 Ser Asn Ala Arg Ala Pro Leu Asp Ile His Ala Ser Leu His Ala Asp -20 -15 -10 -5 Asp Asp Asp Lys Ile Ile Gly Gly Thr Glu Cys Lys Pro His Ser Arg              -1 1 5 10 Pro Tyr Met Ala Tyr Leu Glu Ile Val Thr Ser Asn Gly Pro Ser Lys          15 20 25 Phe Cys Gly Gly Phe Leu Ile Arg Arg Asn Phe Val Leu Thr Ala Ala      30 35 40 His Cys Ala Gly Arg Ser Ile Thr Val Thr Leu Gly Ala His Asn Ile  45 50 55 60 Thr Glu Glu Glu Asp Thr Trp Gln Lys Leu Glu Val Ile Lys Gln Phe                  65 70 75 Arg His Pro Lys Tyr Asn Thr Ser Thr Leu His His Asp Ile Met Leu              80 85 90 Leu Lys Leu Lys Glu Lys Ala Ser Leu Thr Leu Ala Val Gly Thr Leu          95 100 105 Pro Phe Pro Ser Gln Phe Asn Phe Val Pro Pro Gly Arg Met Cys Arg     110 115 120 Val Ala Gly Trp Gly Arg Thr Gly Val Leu Lys Pro Gly Ser Asp Thr 125 130 135 140 Leu Gln Glu Val Lys Leu Arg Leu Met Asp Pro Gln Ala Cys Ser His                 145 150 155 Phe Arg Asp Phe Asp His Asn Leu Gln Leu Cys Val Gly Asn Pro Arg             160 165 170 Lys Thr Lys Ser Ala Phe Lys Gly Asp Ser Gly Gly Pro Leu Leu Cys         175 180 185 Ala Gly Val Ala Gln Gly Ile Val Ser Tyr Gly Arg Ser Asp Ala Lys     190 195 200 Pro Pro Ala Val Phe Thr Arg Ile Ser His Tyr Arg Pro Trp Ile Asn 205 210 215 220 Gln Ile Leu Gln Ala Asn                 225

[Brief description of drawings]

FIG. 1 is a diagram showing the steps for constructing a transfer vector (pYNGsigEKChym) in which sig-EK-chymase DNA is introduced downstream of a polyhedrin promoter in the order of a 30K protein secretion signal sequence and an EK-chymase sequence.

FIG. 2 Transfer vector (pYNGsigE
It is a figure which shows the arrangement | sequence between the 30K protein secretion signal of KChym), and EK- chymase. In the figure, (I) is 30K
It is a figure which shows a protein secretion signal sequence, (II) is an EK- chymase sequence, (a) is a linker sequence, (b) is an enterokinase cleavage sequence, and (c) is a recombinant human chymase sequence.

FIG. 3 is a diagram showing elution curves of recombinant human chymase from silkworm body fluid in each purification step by column chromatography. In the figure, (A) is an elution curve diagram of EK-chymase on a cation exchange column, and (B) is E.
Elution curve diagram of K-chymase by heparin column,
(C) shows the elution curve figure of the recombinant human chymase by the heparin column.

FIG. 4 is a view showing an immunoblot in screening for anti-human chymase monoclonal antibody.

FIG. 5 is a graph showing measured values of purified human blood vessel-derived human chymase treated with untreated and treated with sandwich ELISA using an anti-human chymase monoclonal antibody.

FIG. 6 is a graph showing the measurement values of chymase levels in serum of renal hypertensive patients and healthy subjects, which were measured by a sandwich eliza method using an anti-human chymase monoclonal antibody. that's all

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/543 511 C12N 15/00 ZNAC 33/573 A 33/577 5/00 B (72) Inventor Miyazaki Mizuo 4-5-1 Tomooka-cho, Nagaokakyo-shi, Kyoto (72) Inventor Shinji Takai 1-12-1 Akutagawa-cho, Takatsuki-shi, Osaka F-term (reference) 4B024 AA11 BA07 BA44 CA04 DA02 EA02 GA03 GA11 HA15 4B064 AG27 CA10 CA20 CC24 CE12 DA13 4B065 AA91X AA92X AB05 AC14 BA08 CA25 CA46 4H045 AA11 AA20 AA30 CA40 DA76 DA89 EA50 FA72 GA26

Claims (23)

[Claims] 1. An anti-human chymase monoclonal antibody against a degradation product obtained by enzymatically treating human chymase. 2. The anti-human chymase monoclonal antibody according to claim 1, wherein the enzyme treatment is an enzyme treatment using lysyl endopeptidase. 3. The human anti-human according to claim 1 or 2, wherein the human chymase is a recombinant human chymase obtained by utilizing an insect living body of the Bombycidae or Mothidae or a cultured cell of these insects as a host. Chymase monoclonal antibody. 4. The anti-human chymase monoclonal antibody according to claim 1 or 3, wherein the human chymase has physiological activity. 5. The anti-human chymase monoclonal antibody according to claim 1, which is produced from a hybridoma cell having a deposit number of FERM P-18700. 6. An anti-human chymase monoclonal antibody against recombinant human chymase obtained using an insect living organism of the Bombycidae or Mothidae or a cultured cell of these insects as a host. 7. An animal is immunized with a degradation product obtained by treating human chymase with an enzyme, spleen cells are taken out from the animal, fused with myeloma cells, the resulting hybridoma cells are cultured, and antibodies are collected. A method for obtaining an anti-human chymase monoclonal antibody, comprising: 8. A specimen is pretreated by incubating or heating it in a solution containing a chelating agent, an inhibitor for an enzyme that decomposes chymase, and a surfactant, and then the specimen is immobilized on an anti-human chymase. A method for measuring human chymase, which comprises reacting with a monoclonal antibody, and then reacting the antibody labeled with a labeling substance with an anti-human chymase antibody having a different antigen recognition site to measure the labeled amount of the obtained complex. 9. An anti-human chymase monoclonal antibody immobilized on a solid phase is an anti-human chymase monoclonal antibody against a degradation product obtained by enzymatic treatment of human chymase, or as a host, an organism of an insect of the Bombycidae or Mothidae or culturing of those insects. The method for measuring human chymase according to claim 8, which is an anti-human chymase monoclonal antibody against recombinant human chymase obtained by using cells. 10. The measurement of human chymase according to claim 8, wherein the chelating agent is ethylenediaminetetraacetic acid or ethyleneglycol-bis- (β-aminoethylether) -N, N, N ′, N′-tetraacetic acid. Method. 11. The method for measuring human chymase according to claim 8, wherein the inhibitor of the enzyme degrading chymase is phenylmethylsulfonyl fluoride. 12. The method for measuring human chymase according to claim 8, wherein the surfactant is sodium dodecyl sulfate. 13. The heat insulation or heat treatment is performed at 25 ° C. to 10 ° C.
The method for measuring human chymase according to claim 8, which is performed at a temperature of 0 ° C. 14. A kit for measuring human chymase, which comprises the following components (a) to (c): (A) Anti-human chymase monoclonal antibody immobilized on a solid phase (b) Labeled with a labeling substance, an anti-human chymase antibody different in antigen recognition site from the anti-human chymase monoclonal antibody of component (a) (c) Chelating agent, chymase Pretreatment Agent Containing an Inhibitor for an Enzyme that Decomposes Sulfate and a Surfactant 15. The component (a) antibody is an anti-human chymase monoclonal antibody against a degradation product obtained by enzymatic treatment of human chymase, or an insect living organism of the Bombycidae or Scalidae or a cultured cell of these insects is used as a host. The human chymase assay kit according to claim 14, which is a human chymase monoclonal antibody against the recombinant human chymase obtained. 16. The kit for measuring human chymase according to claim 14, wherein the antibody of component (b) is an anti-human chymase monoclonal antibody. 17. The labeling substance as the component (b) is an enzyme, a radioactive substance, a fluorescent substance or a luminescent substance.
A kit for measuring human chymase according to the item 1. 18. The chelating agent of component (c) is ethylenediaminetetraacetic acid or ethylene glycol-bis- (β
-Aminoethyl ether) -N, N, N ', N'-tetraacetic acid, The kit for measuring human chymase according to claim 14. 19. The kit for measuring human chymase according to claim 14, wherein the inhibitor of the enzyme that decomposes chymase of component (c) is phenylmethylsulfonyl fluoride. 20. The kit for measuring human chymase according to claim 14, wherein the surfactant as the component (c) is sodium dodecyl sulfate. 21. A diagnostic agent for hypertension, which comprises the anti-human chymase monoclonal antibody according to any one of claims 1 to 6. 22. An allergic disease diagnostic agent, comprising the anti-human chymase monoclonal antibody according to any one of claims 1 to 6. 23. A cardiovascular disease diagnostic agent comprising the anti-human chymase monoclonal antibody according to any one of claims 1 to 6.